George J. Haluska

An experimental model for intraamniotic infection and preterm labor in rhesus monkeys

OBJECTIVE Our purpose was to describe the temporal and quantitative relationship between intraamniotic infection and preterm labor in a nonhuman primate model. STUDY DESIGN On day 130 of gestation (term 167 days) four chronically instrumented rhesus monkeys (Macaca mulatta) were infected with an intraamniotic inoculation of 10(6) colony-forming units of group B streptococci. Four additional noninfected monkeys were followed up to spontaneous parturition as controls. Amniotic fluid was serially sampled in all monkeys both before and after inoculation for bacterial growth, tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6, prostaglandin E2, and prostaglandin F2 alpha, and uterine activity was continuously recorded. RESULTS Increases in uterine contractility occurred 28 hours (range 14 to 40 hours) after inoculation and were preceded by increases in amniotic fluid cytokines and prostaglandins. Intraamniotic concentrations of tumor necrosis factor-alpha, interleukin-6, and interleukin-1 beta all rose dramatically 9, 15, and 18 hours after infection and 10 to 20 hours before increases in uterine contractility. In spontaneous parturition only interleukin-6 concentrations rose moderately (from 0.1 to 1.2 ng/ml). Increases in prostaglandin E2 and prostaglandin F2 alpha paralleled those of the cytokines. Peak prostaglandin concentrations in intraamniotic infection exceeded by severalfold concentrations seen in spontaneous parturition (16,046 pg/ml vs 2765 pg/ml for prostaglandin E2, p < 0.05; and 5547 pg/ml vs 708 pg/ml for prostaglandin F2 alpha, p < 0.05). In spite of intraamniotic none of the monkeys were febrile or had peripheral leukocytosis at the onset of labor. CONCLUSION In the rhesus monkey, after intraamniotic infection, there is a predictable and sequential increase in amniotic fluid tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6, followed by increases in prostaglandin E2 and prostaglandin F2 alpha. These increases all occur before an increase in uterine contractility and before clinical signs of infection. Our data provide evidence for a cause-and-effect relationship between intraamniotic infection and preterm labor and support the utility of measuring interleukin-6 or other cytokines in the diagnosis of intraamniotic infection.

Interleukin-Iβ Intra-Amniotic Infusion Induces TUmor Necrosis Factor-α, Prostaglandin Production, and Preterm COntractions in Pregnant Rhesus Monkeys

Objective: To describe the temporal and quantitative consequences of intra-amniotic interleukin-1β infusion in a nonhuman primate model. Methods: On days 128-138 of gestation (term 167 days), four chronically instrumented rhesus monkeys (Macaca mulatta) underwent serial intra-amniotic infusions of 2, 5, and 10-20 μg recombinant human interleukin-1β. Each infusion was for 2 hours, and subsequent infusions were at least 48 hours later. Amniotic fluid was sampled serially both before and after infusion for interleukin-1β, tumor necrosis factor-α (TFN-α), and prostaglandin (PG) E2 and F2α by specific assays, and uterine activity in each monkey was recorded continuously. Results: Intra-amniotic concentrations of interleukin-1β rose dramatically after infusion. This rise was rapidly followed by the appearance of TNF-α in the amniotic cavities of all animals, with maximal levels reached 5 hours after the initiation of the infusion. Both interleukin-1β and TNF-α were rapidly cleared from the amniotic fluid and returned to baseline levels by 24-48 hours. Increases in PGE2 and F2α paralleled those of the two cytokines but remained elevated for the duration of the experiments. The stimulation of uterine contractility from a pre-infusion level of 200 mmHg · seconds/hour to 6000 mmHg · seconds/hour occurred an average of 6-10 hours after interleukin-1β infusion. These stimulations were transient, usually abating by 22 hours after infusion, and did not result in frank labor. Conclusion: In the rhesus monkey, intra-amniotic infusion of interleukin-1β rapidly induces production of intra-amniotic TNF-α as well as PGE2 and F2α, followed by uterine contractility. Uterine activity diminishes as cytokine levels return to pre-infusion levels, even in the presence of elevated intra-amniotic PG levels. Tumor necrosis factor-α may act synergistically with interleukin-1β in the pathophysiology of cytokine-related preterm labor.

Progesterone receptor localization and isoforms in myometrium, decidua, and fetal membranes from rhesus macaques: evidence for functional progesterone withdrawal at parturition.

Objective: It is not known whether withdrawal of progesterone (P) action is a prerequisite for parturition in women or in nonhuman primates because concentrations of circulating progesterone or progesterone receptors (PR) in myometrium and decidua do not decrease before delivery. To examine this potentially important regulatory mechanism, we determined PR isoforms, PR localization, and mRNA in myometrium, decidua, and fetal membranes from rhesus monkeys during pregnancy and in spontaneous labor at term. Methods: Gestational tissues were obtained midpregnancy (day 80-100), late pregnancy (day 130-145), and during spontaneous labor at term (day 161-167). Samples of rhesus monkey myometrium, decidua, chorion-decidua, and amnion were collected and analyzed for total nuclear and cytosolic PR by competitive binding assay. Progesterone receptor isoforms were identified and quantified by Western blot analysis, and PR mRNA was determined by a specific ribonuclease protection assay. Nuclear PR was localized by immunohistochemistry with monoclonal anti-PR (JZB39) after microwave stabilization. Results: Myometrium and decidua showed no change in total PR during pregnancy and labor. Nuclear PR was not detected in fetal membranes by binding assay but was localized in amnion epithelial and mesenchymal cells and in chorion laeve cytotrophoblasts by immunohistochemistry. Staining for PR was substantially less by serial antibody dilution in fetal membranes than in decidua. Message for PR was confirmed in all tissues analyzed. A significant (P < .05) shift in the ratio of PR isoforms (from PR-B dominance at midpregnancy to PR-A dominance in labor) was observed in myometrium but not in decidua. Both PR-A and PR-B isoforms and PR nuclear staining were nearly undetectable in amnion obtained during labor. Conclusion: A shift to PR-A dominance in myometrium at term together with a loss of PR in fetal membranes provides evidence for a functional progesterone withdrawal mechanism, which may facilitate the initiation of parturition in primates.

Induction of interleukin-1 receptor antagonist in rhesus monkeys after intraamniotic infection with group B streptococci or interleukin-1 infusion

OBJECTIVE Interleukin-1 receptor antagonist is a natural inhibitor of interleukin-1, a cytokine implicated in the initiation of preterm labor after intraamniotic infection. The effects of intraamniotic infection and interleukin-1 infusion on the appearance of interleukin-1 receptor antagonist in amniotic fluid and fetal and maternal plasma were assessed with a monkey model. STUDY DESIGN On day 130 of pregnancy four chronically catheterized rhesus macaques received intraamniotic inoculations of group B streptococci, three monkeys received intraamniotic infusions of recombinant human interleukin-1 beta, and three monkeys received buffered saline solution infusions. At timed intervals samples of amniotic fluid, fetal plasma, and maternal plasma were assayed for interleukin-1 beta and interleukin-1 receptor antagonist by immunoassays. Uterine activity was continuously monitored by intraamniotic pressure catheters and by electromyographic activity. RESULTS Interleukin-1 receptor antagonist, but not interleukin-1 beta, was present in the amniotic fluids of all monkeys before intervention. Infection induced the appearance of interleukin-1 beta and an increase in interleukin-1 receptor antagonist in the amniotic fluid. Interleukin-1 beta infusion resulted in a similar increase in the intraamniotic concentration of interleukin-1 receptor antagonist. Both infection and interleukin-1 beta infusion were followed by the transient appearance of interleukin-1 receptor antagonist in the plasma of all fetuses. The subsequent decrease in plasma levels was paralleled by increased amniotic fluid levels of interleukin-1 receptor antagonist. Interleukin-1 beta and interleukin-1 receptor antagonist were not detected in maternal plasma. Both infection and interleukin-1 infusion induced preterm labor in all treated animals. CONCLUSIONS Interleukin-1 receptor antagonist is a normal component of monkey amniotic fluid. Intraamniotic infection or the appearance of interleukin-1 beta in the amniotic fluid results in increased production of interleukin-1 receptor antagonist. Under physiologic conditions interleukin-1 receptor antagonist in amniotic fluid may inhibit interleukin-1-induced preterm labor.

Plasma oxytocin and nocturnal uterine activity: Maternal but not fetal concentrations increase progressively during late pregnancy and delivery in rhesus monkeys*

OBJECTIVE The purpose of the study was to determine whether rising maternal or fetal plasma oxytocin concentrations could be responsible for the increasing levels of nocturnal uterine activity on the nights preceding delivery. STUDY DESIGN Chronically catheterized pregnant rhesus monkeys were exposed to a 16-hour light, 8-hour dark photoperiod (dark 11 PM to 7 AM). Uterine activity and maternal arterial plasma oxytocin concentrations were measured concurrently at weekly intervals in late gestation, on the night preceding term delivery (158 to 167 days, n = 4), and during delivery (149 to 170 days, n = 6). Fetal carotid arterial plasma oxytocin levels were measured during episodes of nocturnal uterine activity in six animals. The effect of oxytocin infusions into the fetus (30 to 480 ng/kg/hr) on uterine activity and on maternal and fetal plasma oxytocin levels was also determined (n = 3). RESULTS Maximal nocturnal oxytocin concentrations in the maternal plasma rose progressively during late gestation from 9.9 +/- 3.5 pg/ml at 130 to 139 days to 28.7 +/- 9.8 pg/ml on the night preceding term delivery (p < 0.005); a significant increase in nocturnal uterine activity accompanied this rise (p < 0.001). Maternal oxytocin concentrations were elevated during labor and increased further at delivery (62.5 +/- 5.5 pg/ml, p < 0.05). There was no increase in fetal plasma oxytocin during nocturnal uterine activity (3.1 +/- 0.2 pg/ml) or during labor. Fetal oxytocin infusions raised fetal plasma oxytocin concentrations sixtyfold but had no effect on maternal plasma oxytocin concentrations or on uterine activity. CONCLUSIONS Elevated maternal plasma oxytocin concentrations are responsible, at least in part, for the increasing magnitude of nocturnal uterine activity episodes as term approaches and for the elevated uterine activity before delivery at night. Fetal plasma oxytocin does not contribute to nocturnal uterine activity or to maternal plasma oxytocin concentrations.

Inhibition and augmentation of progesterone production during pregnancy: Effects on parturition in rhesus monkeys

OBJECTIVES Uterine quiescence during mammalian pregnancy is attributed to progesterone. However. systemic progesterone levels remain elevated in primates before parturition. Epostane, a selective 3beta-hydroxysteroid dehydrogenase inhibitor, and progesterone (with or without epostane) were administered to late pregnant rhesus monkeys to clarify the role of progesterone in primate parturition. STUDY DESIGN On days 122 to 132 of gestation (term 167 days), 11 rhesus monkeys (Macaca mulatta) with timed pregnancies were divided into three treatment groups: (1) epostane alone (10 mg/kg subcutaneously), (2) epostane with progesterone subcutaneously in Silastic silicone rubber capsules, and (3) progesterone implants only with no surgical instrumentation. Maternal and fetal blood and amniotic fluid were sampled for progesterone, estrone, estradiol, cortisol, testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, and amniotic fluid was sampled for prostaglandins E2 and F2alpha. Uterine activity was monitored continuously by electromyography and intraamniotic pressure. Cervical status was assessed by a modified Bishops score. Production of prostaglandins E2 and F2alpha by amnion was determined by tissue superfusion. The group of three noninstrumented monkeys, which received only progesterone Silastic silicone rubber implants subcutaneously at 146 to 148 days, were observed until spontaneous vaginal delivery. RESULTS Epostane reduced maternal and fetal progesterone levels by 75% and 50%, respectively, followed by increased uterine activity and cervical ripening within 24 hours and vaginal delivery within 48 hours. Amniotic fluid progesterone decreased to undetectable levels. Progesterone implants prevented the epostane-induced decrease in maternal and fetal progesterone levels and the associated myometrial and cervical changes until the implants were removed. Alterations in other steroid hormones were consistent with inhibition of 3beta-hydroxysteroid dehydrogenase. Amniotic prostaglandin E2 production was increased sixfold by epostane (p < 0.05) but did not reach the high levels normally seen at spontaneous parturition. Animals that received progesterone implants alone had markedly elevated circulating progesterone concentrations yet were delivered spontaneously at term (range 163 to 167 days). CONCLUSIONS Progesterone withdrawal induces preterm labor and delivery (which can be blocked by progesterone substitution) but exogenous progesterone, even in substantial quantities, does not prevent parturition at term.

New Perspectives on Estrogen, Progesterone, and Oxytocin Action in Primate Parturition

In studying the control of parturition in primates it is logical to concentrate on those endocrine and paracrine regulators which have proven to be important prerequisites for parturition in other species (i.e., estrogens, progesterone, prostaglandins, oxytocin, and cytokines). It has been shown in domestic ruminants that increased estrogen levels and falling progesterone levels at term (dependent upon the fetal adrenal secretion of Cortisol) are pivotal for the initiation of parturition (Liggins et al. 1977; Challis and Olson 1988). Although the rising production of estrogens by the fetoplacental unit provides an attractive mechanism for the endocrine initiation of labor in primates, estrogens likely serve only a permissive function in parturition. There is no evidence for the lowering of progesterone concentrations in the maternal, fetal, or amniotic fluid compartments in higher primates prior to or during labor. However, a prepartum fall in progesterone is such a ubiquitous phenomenon across species that it is still tempting to search for evidence of a localized progesterone withdrawal mechanism in target tissues of primates. Indeed, the recent availability of effective antiprogestins has provided an efficient tool to facilitate such investigation.